Starch in various forms is of great import in the food and paper industry. In the future, starch will also be a great potential for producing polymers which are degradable in nature, e.g. for use as packing material. Many different starch products are known which are produced by derivatization of native starch originating from, inter alia, maize and potato. Starch from potato and maize, respectively, is competing in most market areas.
In the potato tuber, starch is the greatest part of the solid matter. About 1/4 to 1/5 of the starch in potato is amylose, while the remainder of the starch is amylopectin. These two components of the starch have different fields of application, and therefore the possibility of producing either pure amylose or pure amylopectin is most interesting. The two starch components can be produced from common starch, which requires a number of process steps and, consequently, is expensive and complicated.
It has now been proven that by genetic engineering it is possible to modify potato so that the proportion between the two starch components amylose and amylopectin changes in the actual tubers. As a result, a starch quality is obtained which can compete in the areas where potato starch is normally not used today. Starch From such potato which is modified in a genetically engineered manner has great potential as a food additive, since it has not been subjected to any, chemical modification process.
Starch Synthesis PA0 Branching Enzyme in Different Plant Species PA0 Inhibition of Branching Enzyme
The synthesis of starch and the regulation thereof are presently being studied with great interest, both on the level of basic research and for industrial application. Although much is known about the assistance of certain enzymes in the transformation of saccharose into starch, the biosynthesis of starch has not yet been elucidated. By conducting research above all into maize, it has, however, been possible to elucidate part of the routes of synthesis and the enzymes participating in these reactions. The most important starch-synthesizing enzymes for producing the starch granules are starch synthase and branching enzyme. In maize, three forms of starch synthase have so far been demonstrated and studied, two of which are soluble and one is insolubly associated with the starch granules. Also branching enzyme in maize consists of three forms which are probably coded by three different genes.
The starch granules contain a mixture of linear and branched molecules which form the starch components amylose and amylopectin. Amylopectin is produced by interaction between starch synthase and branching enzyme, alpha-1,4-glucane; alpha-1,4-glucane-6-glucosyl transferase (EC 2.4.1.18). Branching enzyme (BE) hydrolyses alpha-1,4 bonds and synthetises alpha-1,6 bonds (Mac Donald & Preiss, 1985; Preiss, 1988).
Endosperm of normal maize contains three forms of BE protein, designated BE I, BE IIa and BE IIb. The mutation amylose extender (amylose extender) inhibits the activity of the enzyme BE Ilb, which results in a reduced content of amylopectin and a corresponding increase of the amylose content. ae endosperm thus has a different proportion of amylose to amylopectin than normal maize, viz. 65:35 instead of 25:75 (De Vries Kuranda, 1987).
Although the similarities between the three enzyme forms are great, each of them has properties in its primary structure which make them unique. The genes for each enzyme form have not been identified so far, but by isolation of cDNA clones for each BE form, each gene can in all probability be characterized.
In normal pea, two forms of branching enzyme (BE) have been identified. A mutation in r locus, which results in a creased pea, affects the activity of BE, thereby inhibiting one enzyme form. This results in a modified composition of the starch with 30% amylopectin and 70% amylose, as compared to the reversed proportion in round normal pea (Smith, 1988).
Branching enzyme (BE) in potato is a monomer protein, i.e. it is a single enzyme form. The molecular weight of potato BE varies between 79 and 103 kD, depending on the purifying process used. There are indications that potato BE should consist of several forms, but presumably several forms are degradation products from the actual protein (Vos-Scheperkeuter, 1989; Blennow & Johansson, 1990).
Peptide sequencing of three BE forms, separated by electrophoresis, has such great homology between the enzyme forms that these are assumed to have the same origin. Serological tests support this assumption, since antisera from the three enzyme forms cross-react with each other.
By inhibiting one of the forms of branching enzyme in maize and pea, the composition of the starch changes so that the content of amylose increases strongly at the sacrifice of the amylopectin production.
In potato, a natural genotype with an increased content of amylose has not been found so far. However, it is possible to reduce the content of BE to a varying extent, which results in the starch in the potato tuber having increased content of amylose as compared to common potato.
The reduction of the formation of enzyme can be accomplished in several ways, e.g. by:
mutagen treatment which results in a modification of the gene sequence coding for the formation of the enzyme
incorporation of a transposon in the gene sequence coding for the enzyme
genetically engineered modification so that the expression of the gene coding for the enzyme is modified by so-called antisense gene inhibition.
FIG. 1 illustrates a specific suppression of normal gene expression in that a complementary antisense nucleotide is allowed to hybridize with mRNA for a target gene. The antisense nucleotide thus is antisense RNA which is transcribed in vivo from a "reversed" gene sequence (Izant, 1989).
By using the antisense technique, various gene functions in plants have been inhibited. The antisense construct for chalcone synthase, polygalacturonase and phosphinotricin acetyltransferase has been used to inhibit the corresponding enzyme in the plant species petunia, tomato and tobacco (Van der Krol et al, 1990; Sheehy et al, 1988; Cornelissen, 1989).
The object of the invention is to provide a varyingly increased amylose production in potato tuber by using antisense gene inhibition.